Cbl-b deficiency diminishes CD28 dependence of T cell activation and enhances susceptibility to autoimmune disease
Yungping J. Chiang1#, Hemanta K. Kole1#, Karen Brown2, Mayumi Naramura1, Shigetomo Fukuhara3, Ren-Ju Hu1, Ihn Kyung Jang1
J. Silvio Gutkind3, Ethan Shevach2, Hua Gu1
1: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, USA
2: National Institutes of Health, Bethesda, MD 20892, USA
3: Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, MD 20892, USA
#: These authors contributed equally to this work.
Correspondence address:
Hua Gu, Ph.D
Laboratory of Immunology
National Institute of Allergy and Infectious Diseases
National Institutes of Health
12441 Parklawn Drive, Rm125
Rockville, MD 20852
USA
Tel: (301) 402 4595
Fax:(301) 594 2522
E-mail:
Supplement Figure legends
Suplement Figure 1 Total tyrosine phosphorylation in Cbl-b-/- T cells. Purified T cells were stimulated as indicated. Total phosphotyrosine was visualized using anti-phosphotyrosine antibody (4G10).
Supplement Figure 2 Actin-polymeryzation of anti-CD-3 antibody-stimulated Cbl/b-/- T cells. Cells were purified as described in the methods. T cells are visualized by green color and F-actin by red color. Arrows indicate the F-actin patches. The white bar in the upper-left panel represents 10 mm scale.
Materials and methods: Cover glass was coated with anti-CD3e antibody (10mg/ml) and treated with poly-L-lysine to enhance cell attachment. Purified T cells were activated on the treated cover glasses for 15-60 minutes at 37oC. Cells were fixed with 3% paraformaldehyde, surfaced stained with biotinylated anti-CD4/CD8 antibodies, followed with streptavidin-FITC. After membrane permeabilization with 0.1% Triton X-100, cells were stained with 1 U/ml Texas Red-conjugated phalloidin (Molecular Probes). Samples were examined under confocal microscopes (Bio Rad and Olympus). Shown are the samples stimulated with anti-CD3e antibody for 45 min.
Supplement Figure 3 Analysis of Vav Cbl-b association in T cells. The association of Vav and Cbl-b is demonstrated by co-immunoprecipitation. Cells were stimulated with anti-CD3e and anti-CD28 antibodies as indicated, and Vav and Cbl-b complexes in the cell lysates were immunoprecipitated using anti-Vav and anti-Cbl-b antibodies, respectively. Co-immunoprecipitated Vav and Cbl-b protein on the same blots were sequentially probed with anti-Cbl-b and anti-Vav antibodies. IP: immunoprecipitation.
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